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Statically adsorbed or covalently coupled capillary coatings are of crucial importance in capillary electrophoresis-mass spectrometry for the separation of peptides and proteins. So far, published coating strategies and commercially available coated capillaries have a limited pH-stability so that the analysis at strongly acidic pH is limited, or harsh rinsing procedures for biological sample analysis cannot be applied. We here present a capillary coating based on Si-C linkages to N-acryloylamido ethoxyethanol (AAEE) with a new synthetic strategy including LiAlH4 surface reaction. We optimized the coating method with emphasis on stability and reproducibility applying harsh rinsing procedures (strong acid, strong base and organic solvent), using the electroosmotic mobility and separation efficiency of tryptic peptides as performance measure. Complete synthesis is performed in less than 2 days for up to 8 capillaries in parallel of more than 16 m total length. Intra- and inter-batch reproducibility were determined regarding electroosmotic mobility, separation efficiency and migration time precision in CE-MS separations of tryptically digested bovine serum albumin. Coating stability towards rinsing with strong acid (1 mol/L HCl), organic solvent (acetonitrile) and strong base (1 mol/L NaOH) was investigated. Outstanding performance was found for single capillaries. However, inter-capillary reproducibility is discussed critically. The new coating was successfully applied for reproducible CE-MS separation of large proteins in diluted serum, medium-sized peptides and small and highly charged polyamines in fish egg extracts using a very acidic background electrolyte containing 0.75 mol/L acetic acid and 0.25 mol/L formic acid (pH 2.2). [ABSTRACT FROM AUTHOR]

Abstract: Two-dimensional electrophoretic separations are one of the most promising tools for the continuously growing needs of different bioanalytical fields such as proteomics and metabolomics. In this work we present the design and the implementation of a two-dimensional electrophoretic separation coupled to mass spectrometry. We started our work studying the sample transfer characteristics of different microfluidic interfaces compatible with capillary coupling for two-dimensional electrophoretic separations. These junctions are aimed at method decoupling and sample transfer in a modular two-dimensional electrophoretic separation system. In order to perform the characterization of the interfaces, we carried out capillary electrophoresis experiments and numerical simulations using three cationic compounds under different flow conditions. The comparison of the experimental and simulation results enables us to clearly define the desirable characteristics of interfaces in order to achieve method orthogonality with lossless sample transfer in a two-dimensional separation system. Finally, we present a glass microfluidic chip as interface for the implementation of a novel hybrid modular system for performing two-dimensional electrophoretic separations involving isotachophoresis and capillary electrophoresis. In this setup we include mass spectrometric and contactless capacitively coupled conductivity detection to monitor the separation process. We demonstrate the ability of the setup to be used as a flexible analysis tool by performing preconcentration, separation, detection and identification of four different human angiotensin peptides. [Copyright &y& Elsevier]

A collaborative study on the robustness and portability of a capillary electrophoresis-mass spectrometry method for peptide mapping was performed by an international team, consisting of 13 independent laboratories from academia and industry. All participants used the same batch of samples, reagents and coated capillaries to run their assays, whereas they utilized the capillary electrophoresis-mass spectrometry equipment available in their laboratories. The equipment used varied in model, type and instrument manufacturer. Furthermore, different types of sheath-flow capillary electrophoresis-mass spectrometry interfaces were used. Migration time, peak height and peak area of ten representative target peptides of trypsin-digested bovine serum albumin were determined by every laboratory on two consecutive days. The data were critically evaluated to identify outliers and final values for means, repeatability (precision within a laboratory) and reproducibility (precision between laboratories) were established. For relative migration time the repeatability was between 0.05 and 0.18% RSD and the reproducibility between 0.14 and 1.3% RSD. For relative peak area repeatability and reproducibility values obtained were 3-12 and 9-29% RSD, respectively. These results demonstrate that capillary electrophoresis-mass spectrometry is robust enough to allow a method transfer across multiple laboratories and should promote a more widespread use of peptide mapping and other capillary electrophoresis-mass spectrometry applications in biopharmaceutical analysis and related fields. [ABSTRACT FROM AUTHOR]